Pakistan Journal of Biochemistry and Biotechnology

Production and Applications of Keratinases in Industry

Sumaira Mazhar, Aisha Waheed Qurash, Tayyaba Latif, Sahar Noor — Wed, 27 Dec 2023 00:00:00 +0000

Keratin is an insoluble protein with fibrous structure. It is mainly found in hair,feathers,nail,wool and horn of various animals. These animal accessories can be utilized as animals feed, amino-acids and fertilizers. This insoluble protein is very difficult to degrade and has extreme stability because it has disulfide bonds, hydrogen and hydrophobic interaction present in it. But keratin can be easily digested by keratinase enzyme. Keratinase is an extracellular enzyme. It also degrades keratin in prokaryotic organisms. Microorganisms that produce keratinase are species of Bacillus, Actinobacteria and fungi. It can also be obtained from Streptomyces, Aspergillus, Fervidobacterium, Xanthomonas, Chryseobacteriumand Vibrio. Keratinases produced by microbes arevery diverse due to their chemical as well as physical properties. It is a vigorous enzyme that can survive broad pH and temperature. Their optimum pH range is neutral to alkaline and their optimum temperature reported is 40°C to 60°C. And thermophiles have also been reported for stable microbial activity. Microbial keratinases are cheaply available as compared to conventional producers for keratinases production. They have gained great significance in biotechnology by acting on strong, inflexible cross linked polypeptide structure of keratin instead of ordinary proteolytic enzyme papain, trypsin or pepsin. They are mostly extracted by degrading keratin as substrate. Keratin substrates include feather, hair, wool,horn and nails. This degradation process is helpful in converting keratinous wastes in to fertilizer and poultry feed. Other renowned biotechnological implementations include removing hair from leather by using keratinase, development of bio-polymer from keratinous fiber, delivering drug, hydrolysis of prion proteins and detergent industry. They are also used in converting biomass in to biofuel that significantly boosts power preservation and recycling. The main problem for producing enzymes on large scale is their costly production procedures. This hurdle is overcome by utilizing keratin waste eg feathers of chicken as fermentation substrate on industrial scale. The benefit of utilizing this left over is extremely cost effective fermentation process of keratinase and the effluents are environmental friendly. Keratinase enzyme has become popular and provided newly effective and efficient way for managing waste products by using them on industrial scales as substrates which gives rise to environmental friendly industries for nonstop development.

A Review on Zoonotic Dermal Infections in Humans

Wed, 27 Dec 2023 00:00:00 +0000

Human skin has a major role in protecting several organs from various microbial attacks. It regulates the body’s temperature and its other normal functions. It is usually possessed by various microbes that may vary in different aspects and impacts on human dermal layer and may lead to serious infections in future. Encounter of human dermal layer to any injury or wound can be a forum for microbes to get entered and cause infection. Most of infections occur due to zoonosis. Anthropozoonosis, zooanthroponosis and amphixenoses are the terms acknowledged for transmit of disease or infections. Zoonosis has three major categories on the basis of type of host, etiological agents and life cycle of pathogens. Each category is further sub-categorized into various possible groups. Domestic animals are generally the primary source for humans to get infected. Human skin is affected by the virus, namely Orf zoonosis having corresponding to the genus Parapoxvirus. It is a major threat to human dermal layer.

In silico identification and characterization of nifH protein of Azospirillum strains isolated from Solanum tuberosum L.

Thu, 28 Dec 2023 00:00:00 +0000

Nitrogen (N) constitutes as a fundamental macronutrient necessary for maintaining plant health, metabolism, and overall survival. However, it exists in the soil in insoluble compound forms that plants cannot directly uptake. Biological N-fixation (BNF) is a fundamental process wherein atmospheric N₂ is reduced to form biologically available ammonium, providing a key N source for plants. The process of BNF is carried out by a diverse yet limited group of microorganisms termed diazotrophs, using the nitrogenase enzyme. This enzyme complex includes two proteins: di-nitrogenase reductase iron protein (nifH), and di-nitrogenase molybdenum iron protein. Due to its significant conservation, the nifH gene serves as a valuable molecular marker for assessing the potential of N-fixation in various microorganisms. Therefore, the current study focused on a comprehensive computational exploration nifH gene and protein of Azospirillum strains (TN03 and TN22). This analysis includes the evaluation of their physicochemical traits, phylogenetic analysis, structural properties (including 3D models), quality assessment of models, and functional annotation analysis using various established bioinformatics tools. The protein of both strains showed an average molecular weight of approximately 14959 and 15313 Da, respectively, exhibited thermal stability and an acidic profile. Furthermore, this theoretical insight could aid researchers in understanding the structure of predicted protein and might facilitate the designing of genetically modified N-fixing bacteria through the design of specific primers.

Protective effect of Helianthus annuus seeds extract against CCl4-induced hepatocellular damage

Fri, 29 Dec 2023 00:00:00 +0000

Carbon tetrachloride (CCl₄) is a hepatotoxin that causes toxicity in animals on its exposure. Hepatotoxins lead to hepatic demage that is treated by generating antioxidant effect. Synthetic and as well as natural drugs are available for this purpose but phyto-based herbal medicines got paramount importance against drug induced hepatotoxicity Current research was conducted to inspect the protective effect of aqueous extract of Helianthus annuus seeds pre-treatment on the carbon tetrachloride-induced hepatotoxicity in Balb C mice. Study covered the valuation of the enzymatic activity such as alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP) and Lactate dehydrogenase (LDH) and other biochemical components like bilirubin, total protein, catalase, glutathione (GSH), malondialdehyde bis-(dimethyl acetal) tetra ammonium (MDA). The induction of carbon tetrachloride (CCl₄) caused rise in plasma ALAT, ASAT, ALP and LDH. Helianthus annuus seeds extract (HA extract) pre-treatment obliterated CCl₄-induced deviations in the activities of these enzymes significantly. Bilirubin level increased whereas total protein contents decreased after the induction of CCl₄ this effect was reversed by HA extract. Induction of CCl₄caused increased in MDA level while decrease in GSH and catalase. Helianthus annuus seeds extract also abolished these changes. Helianthus annuus seeds extract pre-treatment also prevented CCl₄-induced changes in bilirubin and total protein contents. Carbon tetrachloride treatment resulted in huge hepatic damage. This was prevented by Helianthus annuus seeds extract. These results show that Helianthus annuus seeds extract pre-treatment prevented the mice from CCl₄-induced hepatic damage, which visibly shows its defensive effects against hepatic damage.

Phyto-extracts Mediated Biological Control of Citrus Canker

Fri, 29 Dec 2023 00:00:00 +0000

Citrus is second most important fruit crop and has significance economic importance in Pakistan. In Pakistan 2.17 million tonnes citrus was produced on area of 1.94 million acre. Citrus is mostly cultivated in Punjab and affected by different types of plant pathogens. Citrus canker is the most harmful disease among the major diseases of citrus which is caused by Xanthomonas axonopodis pv. citri. The copper based fungicides are mostly employed to manage the citrus canker in the field but excessive use of chemicals have negatively impacted the environment and warrant the need to find out organic solutions therefore the current study was aimed at identifying suitable plants extracts for the control of citrus canker bacterium. Isolation of the bacterium X.axonopodis was done through streaking on agar plate technique. Morphological and bio-chemical tests such as gram staining, KoH test, catalase test and starch hydrolysis test were performed to examine the bacteria X.axonopodispv. citri. Plant extracts were evaluated, in vitro, against the X.axonopodis pv. citri through paper disc method which showed the inhibition zone against the disease. The 100 percent extract of garlic, ginger, aloevera, moringa were used used along with the streptomycin as a positive control. The inhibition zone was 11.67mm and 10.83mm due to garlic and moringa extracts respectively while it was 16.67mm in case of positive control. The ginger and aloevera extracts proved ineffective against the citrus canker bacterium.The efficacy of garlic and moringa extracts were also checked against the X. axonopodis pv. citri under glasshouse conditions. The inoculum was sprayed on Kinnow plants, with three replicates, for each extract and data was recorded 15, 20, 25, 30 and 35 days post inoculation (dpi).The recorded disease severity percentage, after 35dpi, on garlic and moringa treated plants were 9.67% and 14.27% respectively while the 27% disease severity was recorded on control (plants sprayed with autoclaved distilled water). The garlic extract proved to be most effective followed by moringa extract which could be used as a potential organic solution to control citrus canker.

In silico analysis of Bacillus stearothermophilus as a bioindicator for sterilizationco analysis of Bacillus stearothermophilus as a bioindicator for sterilization

Sun, 31 Dec 2023 00:00:00 +0000

To kill every kind of viable bacterial cells, sterilization in food and pharmaceutical industries is essential. In this study, bio indicators are used to validate accurate sterility. Bacillus stearothermophilus is used as a reference standard heat stable bio indicator. A 16S ribosomal RNA ribotyping sequence of Bacillus stearothermophilus is compared with different bacterial bioindicators including Bacillus pumilus, Bacillus atrophaeus, Clostridium sporogenes, and Brevundimonas diminuta. Results showed that Bacillus pumilus has the highest percentage (90.88%) similarity with the Geobacillus stearothermophilus followed by the Bacillus atrophaeus (90.84%), and Clostridium sporogenes (85.35%). The Brevundimonas diminuta was the least similar organism having a percentage similarity of 81.71% with the Geobacillus stearothermophilus. Bacterial spores are used as biological indicators for sterilization because they exhibit resistance to chemical and physical sterilization techniques. The protein common in all bacteria under study is Spo0A that make the bacteria heat resistant. The eradication of all these kind of heat resistant spores needs a high temperature which will also kill other viable cells.

Keywords: Bacillus stearothermophilus; Bacillus pumilus; Bacillus atrophaeus; Clostridium sporogenes and Brevundimonas diminuta; Spo0A protein.

Antibacterial and anti-biofilm profiling of Syzygium Aro-Maticum Plant Extracts against Multi-drug resistant Human Pathogens

Sana Fatima, Saba Saba, Abid Hussain, Laraib Aslam, Azka Naveed — Sun, 31 Dec 2023 00:00:00 +0000

Due to rapidly propagating global resistance to antibiotics/pharmacological agents (One Health Challenge) and costly-expenditure for antibiotic formulation, it is primordial to look for efficient alternatives and ample of evidences document the ability of plant extracts in this regard. This study aimed to evaluate antibacterial and anti-biofilm profiles of Syzygium aromaticum extracts against multidrug-resistant (MDR) pathogens. Sampling of blood and urinary catheters was done from clinical laboratory of Multan Pakistan. A total of 06 pathogenic strains: Streptococcus pyogenes, Staphylococcus saprophyticus, Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, were isolated and confirmed by 16SrRNA Ribotyping. Kirby-Bauer Disc Diffusion method was employed for resistance profiling. Flowers of S. aromaticum were used to prepare 04 different extracts i.e., Methanolic, Ethanolic, Dimethyl sulfoxide and Aqueous. Phytochemical screening showed all extracts positive for Proteins, Carbohydrates, Glycosides, Terpenoids, Saponins and Steroids. MIC and MBC value of all extracts was found 40 µg/ml and 160 µg/ml respectively except Methanolic extract (20 µg/ml and 80 µg/ml respectively). Highest anti-biofilm activity was seen for methanolic extracts. FRAP % was found directly proportional to extract concentration while DDPH % inhibition was: methanolic > ethanolic >Aqueous > DMSO extract. In TLC Analysis, highest R_fvalue was recorded for Methanolic extract. FTIR analysis showed different peaks as: Alkyl halide-698.58/cm, Aromatic group-879/cm, Alcohol-1013.63/cm, Polysaccharides-1045.10/cm, Carboxylic acid-1436.15/cm, Amides-1515.01/cm and Aldehydes/ketones-2975.35/cm. It is concluded that Methanolic extract of Syzygium aromaticum was found to have highest antibacterial, anti-biofilm and antioxidant potential thus its secondary metabolites are cost-effective candidates which can fuel the future pipeline for antibiotic discovery.